Vaccination is mostly by injection. Injection requires complex and expensive logistics. For example, vaccinating large numbers of animals by injection, such as cattle, swine and poultry as well as fish, is either impossible or extremely labour intensive. It would be advantageous in terms of time and expense if the vaccine could be administered, simultaneously, with feed or water to a large number of animals, particularly as repeat doses must usually be given at intervals. Injectable vaccines also generally must be maintained (stored and shipped) at reduced temperature from factory to point of injection. This so-called “cold chain” is a further logistical complexity adding cost.
Apart from the risk of infection and cross-contamination arising from needle use, vaccination by injection is also uncomfortable or painful for the recipient person or animal and alternative administrative routes would be preferable.
Vaccines administered orally or nasally exist and transcutaneous vaccines are in development. However, for a variety of reasons, not all vaccines are available for administration orally, intranasally etc and it would be a significant step forward if more vaccines could be administered other than by injection.
Those oral vaccines that exist are generally live attenuated or killed whole cell preparations which rely on elements of the infectious agent stimulating an immune response through contact with the mucosa in the GI tract or beneath the dermis or other component of the mucosa-associated lymphoid tissue (MALT).
However, many modern vaccines include subunits of the infectious agent which may not by themselves have sufficient immunogenic power to elicit the desired immune response even when injected. Immunogenic power is increased by co-formulation of antigens with immunostimulants such as adjuvants, a phenomenon demonstrated above all for injectable vaccines.
Adjuvant systems to enhance an animal's immune response to a vaccine antigen are well known in the art. Likewise, systems for the delivery of vaccine and drugs to mucosal surfaces are known in the art. Various methods have been described to protect the vaccine antigen and drugs from degradation by stomach acid and digestive enzymes and to adsorb the antigen to the mucosal surface.
Yet mucosal delivery of vaccines has been underutilized because of remaining problems associated with effectively delivering vaccine components to mucosal surfaces and to the underlying mucosal lymphoid tissue, these problems being linked to the formulation complexities of combining various components to be released in active form at the appropriate site. Oral delivery of vaccines giving rise to a mucosal immune response remains a highly desirable goal given the fact that many pathogens invade via mucosal surfaces.
Barriers to achieving effective oral absorption of vaccines include their low permeability across the plasma membrane of intestinal epithelial cells, susceptibility to degradation by peptidases and proteases in the gastrointestinal tract (GIT), and hepatic and biliary clearance of absorbed components from the portal circulation. In the case of protein components, susceptibility to denaturation in the acidic environment of the stomach is also a barrier to oral delivery. Where oral administration of vaccines has successfully elilcited a potentially beneficial immune response, the dose of antigen is often very high. This negatively affects the economics since the cost of goods is high. Dose sparing technologies are therefore required.
Protection of macromolecular vaccine components from acid and enzymatic attack through encapsulation in nano- and microparticulate dosage forms such as polylactide (PLA), polylactide-coglycolide (PLGA), or liposomal-based systems and use of enteric-coated capsules or tablets may offer some protection from enzymatic degradation and acidic attack. However, vaccine encapsulation into particulates such as PLA, PLGA, liposomes, or other particulates may not always be successful as delivery systems owing to their poor absorption into and across intestinal epithelial cells. More generally, even if high quantities of antigen and/or adjuvant can be delivered to the appropriate target (eg M cells and/or Peyer's patches in the GIT), they may not be in appropriate or optimal physico-chemical form (eg appropriately or sufficiently solubilized) to be effective.
Examples of prior art encapsulation and formulation of antigens for oral formulation include the following.
U.S. Pat. No. 5,352,448, Bowersock et al., issued Oct. 4, 1994, describes an oral vaccine formulation for ruminants comprising an antigen in a hydrogel matrix which protects the antigen from degradation in the rumen. The matrix results from cross linking methacrylic acid and methylene bis-acrylamide either in the presence of antigen or for subsequent impregnation by antigen following rehydration. The matrix is preferably pelletized by carrying out polymerisation in a 3-5 mm diameter cylinder followed by cutting the resulting solid cylinder into 3-5 mm thick discs. Ammonium persulfate and sodium bisulfite are exemplified as polymerisation initiators
U.S. Pat. No. 5,674,495, Bowersock et al, issued Oct. 7, 1997, describes a vaccine composition for oral administration comprising an alginate gel in the form of discrete 1-100 μm microparticles made by spraying antigen plus alginate solution into a solution of calcium chloride to effect gelation of the droplets or by adding calcium chloride to an alginate/antigen emulsion. Polymers having functional groups which react with or have an affinity for the alginate surface may be used to coat the particles. Polylysine is an example of such a coating polymer. The microparticles may be formulated by dispersing them in hydrophilic carrier matrices such as classical hydrogels or alginate gel matrices to yield carrier matrix pellets (alginate microparticles in alginate carrier matrix) ranging in size from 2 to 8 mm. The pellets may be coated like the microparticles. This patent also describes a variant in which the microparticles are made with vaccine-containing gelatine in which case gelatine microparticles arise from solidification (via temperature reduction) of an emulsion with an unspecified oil. Stabilization of these gelatine microparticles is achieved by coating with poly-1-lysine.
U.S. Pat. No. 5,500,161, Andrianov et al., issued Mar. 19, 1996, describes microparticles made by dispersing a substantially water insoluble polymer in an aqueous solution in which the substance to be delivered (such as an antigen) is also dissolved, dispersed or suspended, and then coagulating the polymer together with the substance by impact forces (eg shear coagulation) or by chemical coagulation (eg by use of electrolytes, pH changes etc) to form both spherical and non-spherical microparticles. Although the term “microparticle” used in this patent is defined to mean a solid particle ranging in size between 1 and 1000 microns, the largest exemplified microparticles have diameters of 20 microns. According to the patent, microparticles of between 1 and 10 microns are used for certain biological applications such as vaccines.
U.S. Pat. No. 6,015,576, See et al., issued Jan. 18, 2000, describes lyophilized multilamellar liposomes which contain antigen. The liposomes are preferably larger than 20 nm and smaller than 20 μm to ensure adequate processing by macrophages. The liposome preparation is lyophilised before being packaged for oral administration as a pill or capsule which may be enteric coated.
U.S. Pat. No. 5,811,128, Tice et al., issued Sep. 22, 1998, describes compositions for delivering a bioactive agent (especially vaccine antigens) to an animal entailing the steps of encapsulating effective amounts of the agent in a biocompatible excipient such as poly (DL-lactide-co-glycolide), to form microcapsules having a size less than approximately ten micrometers. They are made by dispersing an aqueous solution of antigen in polymer solution in methylene chloride. This polymer solution is then added to an aqueous poly(vinyl alcohol) solution to form an oil-in-water emulsion from which the microcapsules are collected by centriguation and freeze drying. Suspensions of the microcapsules were administered using an intubation needle.
Chitosan micropartices may also be useful for oral vaccines. Van de Lubben et al in J. Drug Target, 2002, describe 1.7 μm chitosan microparticles being transported by M cells in an ex-vivo human cell model.
Li et al. in BMC Biotechnology, 2008, describe alginate-coated chitosan microparticles (initially 300 nm in mean size) for vaccine delivery. Antigen (bovine serum albumin) was loaded on the chitosan microparticles by incubating the microsparticles in albumin (resulting mean size 404 nm). Drops of a suspension of antigen-loaded microparticles were then introduced into sodium alginate solution to yield alginate-coated (antigen-loaded) microparticles which were re-dispersed into calcium chloride to crosslink the alginate layer on the surface of the microparticles (resulting mean size: 1324 nm).
One approach to enhance drug and particulate delivery into and/or across the intestinal epithelial barrier is to target particulate formulations to receptor sites of the intestine. For example, Higgins et al. in Pharmaceutical Research Vol 21, 2004, employ small organic peptido-mimetics of the glycoprotein UEA-1 lectin to target M-cells. These mimetics are adsorbed to fluorescein isothiocyante-loaded streptavidin polystyrene particles with a diameter of 0.289 μm using biotinylated peptides. Roth-Walter et al. in Vaccine, Vol 23, 2005, use 1-3 μm sized vaccine-loaded poly(D,L-lactic-co-glycolic acid) microspheres functionalized with alpha-L-fucose specific Aleuria aurantia lectin (AAL). Both these documents (Higgins et al. and Roth-Walter et al.) are incorporated herein by reference in their entirety.
Polylactide-coglycolide (PLG) systems, such as those described above, have been tested as potentially useful in both injectable and oral vaccine formulations as described by Vajdy et al. in Immunology and Cell Biology, Vol. 82, 2004, the entirety of which is incorporated herein by reference. This paper also describes use of emulsions as adjuvants for injectable vaccines and points out that potential toxicity of emulsion components constrains their use although adjuvants such as MF59 (squalene oil-in-water emulsion) by Chiron/Novartis and AS03 ie. squalene (10.68 milligrams), DL-α-tocopherol (11.86 milligrams) and polysorbate 80 (4.85 milligrams) by GSK have been registered e.g. as components of injectable flu vaccines. See for example the product information for Preprandrix™ on EMEA's website.
It would be desirable to be able to use emulsions as components of (or as adjuvants in) oral vaccine formulations.
U.S. Pat. No. 5,961,970 (Lowell et al) describes vaccine adjuvant compositions in the form of an emulsion of a plurality of submicron oil-in-water droplets having a particle size in the range of between about 30 nm to about 500 nm to effect enhanced immunogenicity of antigens incorporated intrinsically or extrinsically into the droplets. To achieve mucosal immunity, the emulsion may also comprise a mucoadhesive macromolecule. To facilitate intestinal uptake, the emulsions may be encapsulated in gelatin capsules or otherwise enterocoated to prevent their exposure to gastric fluids when the oral route of administration is selected. Furthermore, the emulsions may be lyophilized prior to their encapsulation in order to achieve added stability of the antigen. The emulsion particles have a hydrophobic core comprising a lipid or lipid-like composition (eg MCT) and are stabilized with amphiphilic and/or non-ionic surfactants which may be a natural biologically compatible surfactant such as phospholipid (e.g., lecithin) or a pharmaceutically acceptable non-natural surfactant such as TWEEN-80. The surfactant assists in maintaining particles within the desired size range and preventing their aggregation.
US patent application 2001/0043949 (Delgado et al) describes a microparticulate composition comprising a biodegradable synthetic polymer microparticle, an antigen and an enteric polymer forming a coating on the microparticle surface.
Oral vaccines should preferably be solid or substantially solid in order to facilitate processing and storage, enhance stability (especially antigen stability) and to avoid the need for cold chain handling—liquids tend to be less stable than solids while liquids require more elaborate filling and containment in vessels, vials, syringes etc than do solid dose vaccines.
PCT application WO/2008/122967 (Sigmoid Pharma Limited) describes an oral composition comprising minicapsules having a liquid, semi-solid, or solid core and FIG. 2 therein is a schematic of a semi-solid- or solid-filled minicapsule/minisphere wherein the active principle is solubilised or in a suspension form, with controlled release polymer coatings. Example 20 describes beads of an extruded emulsion drug suspension made from mixing an aqueous solution with an oil solution made up of squalene (a natural unsaturated hydrocarbon), Gelucire 44/14 and Labrafil MS1944 CS. The water-soluble active principle hydralazine is in the aqueous phase and the oil phase is 1.12 dry weight % of the formulation.
Dry powder vaccines exist for intra-nasal delivery as described for example by Garmise et al in AAP PharmSciTech, Vol. 7, 2006. Solid dose vaccines for oral delivery also exist but are rare with the principal example being cholera vaccine tablets in which heat-killed whole cells of V. cholera are tabletted using traditional techniques.
If it is desired to incorporate surfactants in a minicapsule or minisphere formulation of an emulsion, a particular challenge arises. The need for surface tension to create and maintain capsules during manufacture can preclude or limit use of surfactants as the reduction in surface tension caused by the surfactant can destroy the integrity of the capsule or cause a more monolithic format where for example a shell or capsular layer is desired. Thus it can be difficult to formulate liquid, emulsified or pre-solubilized active principles with surfactants.
It may be desirable that vaccine compositions release active principles (eg antigens and adjuvants) in the colon following oral administration. Such colon-specific delivery systems must prevent the release of the active principles in the upper part of the GIT yet release them on reaching the colon. In the art of pharmaceutical delivery, there are a number of formulation approaches including pH and time-dependent polymer-mediated technologies. However, while variations in pH between the small intestine and the colon are well documented, the differences can be small and can vary between individuals. This can make pH-dependent systems unreliable in obtaining a predictable drug release profile. Time-dependent systems depend on the transit time of the delivery system in the GIT. A major limitation with these systems is that in vivo variation in the small intestinal transit time may lead to release of the active principles in the small intestine (too early) or in the terminal part of the colon (too late). The patho-physiological state of the individual recipient of such oral drug delivery systems also has a significant effect on the performance of these time-dependent systems—patients with irritable bowel syndrome and inflammatory bowel disease (including Crohn's disease and ulcerative colitis) exhibit accelerated transit through the colon. Independently of these considerations, the size of the dosage form at the point of entry into the small intestine (pylorus) can have a significant effect on GI transit time and/or variability of response.
The intestinal mucosal immune network has evolved an ability to maintain relative unresponsiveness or tolerance (or “oral tolerance”) to a wide array of antigens derived from dietary sources and commensal bacteria. According to Friedman et al., PNAS USA 1994; 91:6688-92, oral tolerance is mediated by the generation of active cellular suppression or clonal anergy and the determining factor is the dose of antigen fed orally. Oral tolerance is dose-specific and loss of tolerance occurs with increased dosages according to Nagler-Anderson et al., PNAS USA 1986; 83:7443-6. Low dose of antigen administration favours the induction of active cellular regulation according to Chen et al., 1994 Science; 265:1237-1240. Higher doses favour the induction of clonal anergy or deletion according to Chen et al., 1995 Nature; 376:177-180. In a particular study, high doses of myelin basic protein (MBP) to mice whose T-cells carry a T-cell receptor (TCR) specific for MBP resulted in T-cell activation and receptor down modulation (Benson et al., 2000 J Clin Invest, 106:1031-1038). Additionally, oral tolerance can be enhanced by feeding immune adjuvants such as lipopolysaccharide or cholera toxin subunit B, which appear to stimulate additional populations of cells to down-regulate immune responses (Khoury et al., J Exp Med 1992; 176:1355-64).
Oral tolerance has been shown to prevent or treat a variety of T-cell mediated autoimmune disorders. For example, in a double-blind pilot trial involving 30 patients with multiple sclerosis, oral administration of bovine myelin antigens decreased the number of T-cells that reacted with myelin basic protein, with no measurable toxicity (Werner et al., Science 1993; 259:1321-4). Trentham et al demonstrated clinical efficacy of oral tolerance by feeding type II collagen to 60 patients with severe, active rheumatoid arthritis (Trentham et al., Science 1993; 261:1727-30). In an animal model of trinitrobenzene sulfonic acid (TNBS), Th1-mediated colitis, it was reported that feeding colonic extracts haptenated with TNBS prevented the development of mucosal inflammation (Neurath et al., J Exp Med 1996; 183:2605-16). In a Phase I study to evaluate the safety and efficacy of autologous colonic protein extract feeding for the treatment of moderate-to-severe Crohn's Disease, Margalit et al. demonstrated safety and induced remission in 7 out of 10 subjects (Am J Gastroenterol 2006; 101). Other animal disease models, including stroke, Alzheimer's disease and atherosclerosis as well as type 1 diabetes have responded to mucosal administration of antigens.
Various responses are induced or suppressed during oral tolerance beginning when antigen first encounters gut-associated lymphoid tissue (GALT), a well developed immune network consisting of lymphoid nodules (Peyer's Patches), epithelial villi, intraepithelial lymphocytes, and other lymphocytes scattered throughout the lamina propria in the GIT.
More generally, the lamina propria is a constituent of the moist linings (mucous membranes or mucosa) which line various tubes in the body (such as the respiratory tract, the gastrointestinal tract, and the urogenital tract). Thus, the lamina propria (more correctly lamina propria mucosae) is a thin layer of loose connective tissue which lies beneath the epithelium and together with the epithelium constitutes the mucosa. The lamina propria contains capillaries and a central lacteal (lymph vessel) in the small intestine, as well as lymphoid tissue. Lamina propria also contains glands with the ducts opening on to the mucosal epithelium, that secrete mucus and serous secretions.
Antigens may act directly at the level of the GALT or may exert their effects after absorption. Oral tolerance and mucosal immunization form part of an immunologic continuum related to antigen presenting cell interactions with T cells in the GALT. Distinct sections of the GI tract can be distinguished. The rectum/colon is a mix of immune inductive (organised lymphoid tissues) and effector sites (diffuse lamina propria) whereas the jejunum contains almost no immune inductive sites. This is reflected in the lymphoid composition of each tissue: the jejunum contains mostly memory CD4+ T cells, while the colon contains a larger proportion of naïve CD4+ T cells (Veazey and Lackner, 2006; PLoS Medicine; 3:12-2188-9).
In acute HIV infection, a rapid and profound loss of CD4+ CCR5+ T cells occurs within days of infection, whereas peripheral lymphoid tissues such as blood and lymph nodes, which harbour mainly naïve CD4+ T cells, are less severely affected (Brenchley et al., 2004 J Exp Med 200:749-759). Mehandru et al. studying lymphocyte populations from the intestine and peripheral blood obtained from recently HIV-infected patients as well as uninfected volunteers demonstrated that most patients who initiate high activity anti-retroviral therapy (ART) as early as possible after HIV infection still do not experience complete restoration of intestinal CD4+ T cells to baseline levels, regardless of the duration of therapy. Instead, HIV infection results in a continuous state of activation in the intestinal immune system that is not reflected in peripheral lymphoid tissues (Mehandru et al., 2006; PLoS Med 3(12): e484). The data from Mehandru et al. provide evidence that intestinal inflammation and continual infection, destruction, and turnover of CD4+ T cells occur in patients on ART. This would suggest that drugs with better intestinal tissue distribution, together with, perhaps, mechanisms to reduce or prevent immune activation in mucosal tissues may more effectively combat HIV infection.
A number of colon targeted delivery systems have been investigated. These systems include: intestinal pressure-controlled colon delivery capsules which rely on peristaltic waves occurring in the colon but not in the stomach and small intestine; combination of pH-sensitive polymer coatings (remaining intact in the upper GIT) with a coating of polysaccharides degradable only by bacteria found in the colon; pectin and galatomannan coating, degraded by colonic bacteria; and azo hydrogels progressively degraded by azoreductase produced by colonic bacteria. The preceding four systems are reviewed by Yang et al., International Journal of Pharmaceutics 235 (2002) 1-15, the entirety of which is incorporated herein by reference. Polysaccharide based delivery systems are of particular interest—see e.g. Kosaraju, Critical Reviews in Food Science and Nutrition, 45:251-258 (2005) the entirety of which is incorporated herein by reference. Nevertheless, for systems solely reliant on specific enzymatic activity in the colon, disease state can once again cause variability in the drug release profile as a result of pathological derangements in colonic flora (eg resulting from pH changes and changing amounts/activity of bacterial enzymes).
It is not unusual that multiple immunizations are required for many vaccines to be successful. For paediatric population, up to five immunizations may be needed, as is the case for diphtheria, tetanus, and pertussis (DTP) vaccine, which is given three times during the first six months after birth, followed by a fourth dose in the second year of life, and a final boost between four and six years of age. However, some of the vaccines need additional boosts even in adults who have already received the complete immunization series, for example, the tetanus-diphtheria (Td) vaccine, for which a boost is recommended every 10 years throughout a person's lifespan. The “prime-boost” principle applies to live attenuated vaccines (e.g. oral polio vaccine), inactivated vaccines (e.g. hepatitis A vaccine), recombinant protein subunit vaccines (e.g. hepatitis B vaccine), and polysaccharide vaccines (e.g. Haemophilus influenzae type b vaccine). For these vaccines, the prime-boost is ‘homologous’ because the same vaccines given in the earlier priming immunizations are used for subsequent boost immunizations. For more detailed discussion, see Curr Opin Immunol. 2009 June; 21(3):346-51. Epub 2009 Jun. 6, the entirety of which is incorporated herein by reference.
Over the past decade, studies have shown that prime-boost immunizations can be given with unmatched vaccine delivery methods while using the same antigen, in a ‘heterologous’ prime-boost format. The most interesting and unexpected finding is that, in many cases, heterologous prime-boost is more effective than the ‘homologous’ prime-boost approach. The rapid progress of novel vaccination approaches, such as DNA vaccines and viral vector-based vaccines, has certainly further expanded the scope of heterologous prime-boost vaccination and frequently used heterologous prime-boost vaccinations include DNA priming followed by boosting with recombinant protein, inactivated vaccine, viral vectors and BCG; priming with viral vector followed by boosting with recombinant protein; and priming with BCG followed by boosting with viral vector.
It is known that effective B cell-mediated immunity and antibody responses often require help from CD4+ T cells. It is thought that a distinct CD4+ effector T cell subset, called T follicular helper (TFH) cells, provides this help. According to Johnston et al's work published online in Science on Jul. 16, 2009 (DOI: 10.1126/science.1175870) the entirety of which is incorporated herein by reference, expression of the transcription factor Bcl6 in CD4+ T cells is both necessary and sufficient for in vivo TFH differentiation and T cell help to B cells in mice. These researches also state that in contrast, the transcription factor Blimp-1, an antagonist of Bcl6, inhibits TFH differentiation and help, thereby preventing B cell germinal center and antibody responses. Thus TFH cells are required for proper B cell responses in vivo and that Bcl6 and Blimp-1 play central yet opposing roles in TFH differentiation.